Application of corn ZmCHI gene in regulating and controlling resistance of plants to pratylenchus heterodera

Abstract

The invention discloses application of a corn ZmCHI gene in regulating and controlling the resistance of plants to pratylenchus brachypus, and belongs to the technical field of prevention and control of plant pathogenic nematodes. The nucleotide sequence of the ZmCHI gene is as shown in SEQ ID NO.4, and the amino acid sequence of the encoded protein of the ZmCHI gene is as shown in SEQ ID NO.3. The invention finds that the ZmCHI expression quantity is obviously reduced by about 45% by silencing the corn ZmCHI gene through virus-mediated VIGS, the rhizosphere infection insect quantity of an RNAi-ZmCHI corn plant is increased by about 13%, and the growth amounts such as fresh weight and root weight of the overground part of the corn are obviously reduced. The ZmCHI gene is overexpressed, the expression quantity of ZmCHI is remarkably improved, the quantity of rhizosphere infected insects of ZmCHI overexpressed corn plants is remarkably reduced compared with that of a control group, the increment of fresh weight, root weight and the like of the overground part of the corn is remarkably improved, and the resistance of the corn to pratylenchus coffee is remarkably enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of nematode control, and particularly to the application of maize ZmCHI gene in regulating plant resistance to Pratylenchus coffeae. Background Art

[0002] Pratylenchus coffeae is a plant pathogenic nematode with important economic harm in agricultural production. It causes serious damage to the roots of various crops, resulting in significant economic losses to global agricultural production. Pratylenchus nematodes infect root tissues, leading to stunted root development, root rot, growth retardation and plant death of crops. The mechanical damage caused by nematode stylets to crop roots makes plants more susceptible to other pathogens, such as bacteria, fungi and viruses, thus increasing the risk of plants suffering from other diseases and posing a greater challenge to the current global food crisis. Therefore, in-depth research on the infection mechanism and prevention and control of Pratylenchus coffeae has great theoretical guiding and practical significance.

[0003] In recent years, many research results have been obtained in the study of plant parasitic nematodes, which have deepened the understanding of the infection and pathogenesis of Pratylenchus nematodes from many aspects. However, the infection mechanism of Pratylenchus coffeae is still unclear, and the related research on the interaction with hosts to regulate the host plant immune response and promote parasitism is very scarce. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of maize ZmCHI gene in regulating plant resistance to Pratylenchus nematodes to solve the problems existing in the above-mentioned prior art. This study found that β-1,4-endoglucanase PcENG2 interacted with maize chitinase (ZmCHI), promoting the infection and parasitism of Pratylenchus coffeae on maize. This chapter takes the interaction between PcENG2 and ZmCHI as the entry point to deeply analyze the interaction mechanism between the two and the disease-resistant pathway of ZmCHI.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is the application of ZmCHI gene in regulating plant resistance to Pratylenchus nematodes, and the nucleotide sequence of ZmCHI gene is shown in SEQ ID NO.4.

[0007] Another technical solution of the present invention is a method for improving plant resistance to Pratylenchus nematodes, which improves the resistance of the plant to Pratylenchus nematodes by overexpressing the ZmCHI gene in the plant.

[0008] Another technical solution of the present invention is the application of a recombinant vector, expression cassette or transgenic cell line containing ZmCHI gene in improving plant resistance to Pratylenchus nematodes.

[0009] The fourth technical solution of the present invention is the application of ZmCHI protein in regulating the resistance of plants to Pratylenchus coffeae, and the amino acid sequence of ZmCHI protein is shown in SEQ ID NO.3.

[0010] The fifth technical solution of the present invention is the application of ZmCHI protein in inhibiting the hatching of Pratylenchus coffeae eggs.

[0011] Based on the above technical solutions, the present invention has the following technical effects:

[0012] The present invention found that the results of virus-mediated RNAi in maize showed that the RNAi-ZmCHI maize plants had weakened resistance to Pratylenchus coffeae, and the expression level of ZmCHI decreased significantly by about 45% after transforming maize with Agrobacterium tumefaciens carrying TRV-ZmCHI. Statistical results of the test after inoculating nematodes on maize roots in different treatment groups for 60 days showed that the number of nematodes infecting the rhizosphere of RNAi-ZmCHI maize plants increased by about 13%, and the growth parameters such as the fresh weight of the above-ground part and root weight of maize were significantly lower than those of the control group.

[0013] The inventors constructed ZmCHI overexpressing maize plants, with wild-type maize plants as the control group. The results showed that the expression level of ZmCHI increased significantly. Statistical results of the test after inoculating nematodes on maize roots in different treatment groups for 60 days showed that the number of nematodes infecting the rhizosphere of ZmCHI overexpressing maize plants decreased, and the growth parameters such as the fresh weight of the above-ground part and root weight of maize increased significantly, and the resistance of maize to Pratylenchus coffeae was significantly enhanced. Therefore, maize ZmCHI plays an important role in inhibiting nematode infection and enhancing host resistance.

[0014] The present invention also found that ZmCHI-GST could significantly inhibit the hatching of Pratylenchus coffeae eggs, and the hatching rate of eggs decreased by about 25.6% compared with the control group after co-incubation for 24 h and 48 h.

[0015] The above results confirmed that ZmCHI is an important regulatory factor, which plays an important role in regulating maize resistance to Pratylenchus coffeae infection, pathogenesis and egg hatching. The results of this study provide a theoretical basis for maize breeding for resistance to Pratylenchus coffeae, and also provide potential control targets for the development of new control drugs against Pratylenchus coffeae. Brief Description of the Drawings

[0016] Figure 1 RNAi-PcENG2 mediated by maize. Among them, A: Maize-mediated RNAi-PcENG2, Agrobacterium tumefaciens carrying pTRV2 was used as the blank control, and Agrobacterium tumefaciens carrying pTRV-ZmPDS showing photo-bleaching phenotype was used as the positive control, 10 dpa; B: PCR detection of positive seedlings of TRV2-PcENG2.

[0017] Figure 2Staining coffee root-lesion nematodes in maize roots. Among them, A: WT; B: TRV2-GFP; C: TRV2-empty; D: TRV2-PcENG2; Scale bar: 500 μm.

[0018] Figure 3 Effect of maize-mediated RNAi-PcENG2 on nematode pathogenicity. Among them, A: PcENG2 expression level; B: Nematode quantity in maize roots at 60 dpi after inoculation; C: Root weight of maize at 60 dpi after inoculation; D: Shoot weight of maize at 60 dpi after inoculation.

[0019] Figure 4 Prokaryotic expression and WB detection of PcENG2. Among them, A: WB detection of His-PcENG2; B: WB detection of His-PcENG2 at different concentrations; 1: 0.4 mg / mL BSA; 2: Marker; 3: Protein sample diluted 40-fold; 4: Protein sample diluted 20-fold.

[0020] Figure 5 Immunofluorescence detection of PcENG2 in maize roots. Among them, A-B: Immunofluorescence localization of anti-PcENG2 antibody in sections of maize roots infected by nematodes; C: Hybridization of maize sections with pre-immune rabbit serum against PcENG2 antigen, scale bar = 100 μm; D: Hybridization of maize sections with CY3-labeled secondary antibody, scale bars in A, B, and D = 200 μm; Red arrow: Fluorescent signal of PcENG2 secreted by coffee root-lesion nematodes.

[0021] Figure 6 Immunofluorescence scanning of the action site of PcENG2 in maize roots. Among them, A-B: Distribution of PcENG2 protein in maize roots, indicated by white arrows; Scale bar = 100 μm.

[0022] Figure 7 Signal peptide prediction of PcENG2.

[0023] Figure 8 Verification of signal peptide in the yeast system.

[0024] Figure 9 PcENG2 inhibits the burst of reactive oxygen species (ROS).

[0025] Figure 10 PcENG2 inhibits programmed cell death in Nicotiana benthamiana cells.

[0026] Figure 11 PcENG2 is involved in regulating the expression of defense-related genes.

[0027] Figure 12For the screening of PcENG2 interacting proteins. Among them, A: Screening of positive yeast monoclonal colonies; B: PCR detection of positive yeast monoclonal bacterial liquid.

[0028] Figure 13 For the yeast interaction verification of ZmCHI and PcENG2. Among them, SD-WL: Synthetic dropout medium without tryptophan and leucine; SD-WLHA: Synthetic dropout medium without tryptophan, leucine, histidine, and adenine; AD-T + BD-Lam: Negative control; AD-T + BD-53: Positive control.

[0029] Figure 14 For the verification of the interaction between ZmCHI and PcENG2 by Pull-down.

[0030] Figure 15 For the verification of the interaction between ZmCHI and PcENG2 by luciferase complementation.

[0031] Figure 16 For the BIFC interaction verification of PcENG2 and ZmCHI. Among them, Merged: Merged channel; YFP: Yellow fluorescent protein channel; Bright: Bright field; Scale bar = 20 μm.

[0032] Figure 17 For the determination of the resistance of TRV-mediated maize RNAi-ZmCHI to Pratylenchus coffeae. Among them, A: Detection of the expression level of maize ZmCHI gene; B: Total amount of Pratylenchus coffeae in the rhizosphere of maize at 60 days after inoculation; C: Fresh weight of maize roots at 60 days after inoculation; D: Fresh weight of the above-ground part of maize at 60 days after inoculation.

[0033] Figure 18 For the determination of the broad-spectrum resistance of TRV-mediated RNAi-ZmCHI maize to Pratylenchus spp. After T-test, asterisks represent significant differences (*, P < 0.05).

[0034] Figure 19 For the effect of ZmCHI on the morphology of Pratylenchus eggs. PBS: Control group; ZmCHI: Group treated with only ZmCHI protein; ZmCHI + PcENG2: Group treated with both ZmCHI and PcENG2.

[0035] Figure 20 For the effect of ZmCHI on the hatching of Pratylenchus eggs after 24 and 48 hours of treatment. After T-test, asterisks represent significant differences (**, P < 0.01).

[0036] Figure 21 For the determination of ZmCHI enzyme activity. After T-test, asterisks represent significant differences (**, P < 0.01). Specific implementation manners

[0037] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be construed as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and implementation schemes of the present invention.

[0038] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art. The reagents or raw materials used, unless otherwise specified, are all purchased from commercial channels or are publicly available.

[0039] The embodiments of the present invention provide the application of the ZmCHI gene in regulating the resistance of plants to Pratylenchus nematodes. The nucleotide sequence of the ZmCHI gene is shown in SEQ ID NO.4.

[0040] In some specific implementation schemes, overexpressing the ZmCHI gene can improve the resistance of plants to Pratylenchus nematodes; silencing or knocking out the ZmCHI gene can reduce the resistance of plants to Pratylenchus nematodes.

[0041] In some specific implementation schemes, the plants include maize.

[0042] In some specific implementation schemes, the Pratylenchus nematodes include Pratylenchus coffeae, Pratylenchus zeae, Pratylenchus scribneri, and Pratylenchus penetrans.

[0043] The embodiments of the present invention also provide a method for improving the resistance of plants to Pratylenchus nematodes. By overexpressing the ZmCHI gene in plants, the resistance of the plants to Pratylenchus nematodes can be improved.

[0044] In some specific implementation schemes, the methods for overexpressing the ZmCHI gene in plants are selected from at least one of the following methods:

[0045] 1) By introducing a plasmid with the ZmCHI gene;

[0046] 2) By increasing the copy number of the ZmCHI gene on the plant chromosome;

[0047] 3) By changing the promoter sequence of the ZmCHI gene on the plant chromosome;

[0048] 4) By operably linking a strong promoter to the ZmCHI gene;

[0049] 5) By introducing an enhancer.

[0050] The embodiments of the present invention also provide the application of a recombinant vector, expression cassette, or transgenic cell line containing the ZmCHI gene in improving the resistance of plants to Pratylenchus nematodes.

[0051] The embodiments of the present invention also provide the application of the ZmCHI protein in regulating the resistance of plants to Pratylenchus nematodes. The amino acid sequence of the ZmCHI protein is shown in SEQ ID NO.3.

[0052] The embodiment of the present invention also provides the application of ZmCHI protein in inhibiting the hatching of Pratylenchus coffeae eggs.

[0053] Example 1

[0054] 1 Materials

[0055] 1.1 Tested nematode populations: The Pratylenchus coffeae, Pratylenchus zeae, Pratylenchus scribneri, and Pratylenchus penetrans used in the experiment were all cultured and preserved in this laboratory.

[0056] 1.2 Tested plant materials: Nicotiana benthamiana: Cultured in a 25°C greenhouse until 4 weeks old; Maize: The variety is Zhengdan 958, cultured in a 28°C greenhouse, and cultivated to the three-leaf and one-core stage at the seedling stage for standby.

[0057] 1.3 Preparation of main culture media:

[0058] (1) Nematode fixative: Measure 50 mL of 3.7% paraformaldehyde in a large beaker, slowly add 50 mL of 1×PBS buffer in a fume hood, and store at room temperature in the dark.

[0059] (2) LB liquid medium: Weigh 5 g of Tryptone, 5 g of NaCl, and 5 g of Yeast extract in a beaker, add 400 mL of sterile water, adjust the pH to 7.4 with 5M NaOH, make up the volume to 500 mL, sterilize at 121°C and 0.1 MPa for 30 min, and then store for later use.

[0060] (3) MMA solution: 10 mM CaCl2, 10 mM MES, 0.1 mM acetosyringone, made up to 200 mL with sterile deionized water, stored at 4°C in the dark;

[0061] 2 Test methods

[0062] 2.1 Nematode culture and plant sample treatment

[0063] Prepare carrot callus according to the method of Reise et al. The tested Pratylenchus coffeae population was disinfected overnight with 0.3% streptomycin sulfate solution, washed repeatedly with sterile water, and then inoculated onto carrot culture dishes in a laminar flow hood and cultured in the dark at 25°C (Reise et al., 1987). The nematodes on the roots were separated using the modified Baermann funnel method, mainly referring to the method of Kaplan et al. and making improvements (Kaplan et al., 1997). Maize seeds were surface-sterilized with 75% ethanol and then placed in a culture dish containing sterile wet filter paper for germination for 2 days. The seedlings were transplanted into flower pots containing soil and substrate (1:1), and the light setting was light:dark = 16 h:8 h, and the temperature was 28°C. Nicotiana benthamiana grew under a 16 h photoperiod at 25°C.

[0064] 2.2 RNA extraction of samples

[0065] Extract the RNA of Pratylenchus coffeae and store it in a cryogenic refrigerator for later use.

[0066] 2.3 RT-qPCR and reaction system

[0067] AdvanceFast One-step RT-gDNA Digestion SuperMix for qPCR Instruction Manual Operations (All operations are carried out on ice. The reaction program settings refer to the instruction manual of the kit. The operation steps are as follows:

[0068] (1) The reverse transcription reaction system is shown in Table 1.

[0069] Table 1

[0070] (2)

[0072] The qRT-PCR reaction system is shown in Table 2.

[0073] Table 2

[0074]

[0075] 2.4 Paraffin Sections and Immunofluorescence of Maize Roots

[0076] Paraffin section is a method widely used in plant pathological tissue analysis. After rinsing the maize roots infected with Pratylenchus coffeae, cut them into small sections about 1 cm long. The specific steps are as follows:

[0077] (1) Immediately after sampling, soak in FAA fixative and place in a 4°C refrigerator for 48 h for refrigeration treatment;

[0078] (2) After taking out, rinse thoroughly with ultrapure water and soak in PBS buffer for 30 min;

[0079] (3) Place the samples in 50%, 75%, 95% and 100% ethanol for gradient dehydration, with each dehydration duration being 2 h;

[0080] (4) Transparify the samples (ethanol:xylene = 1:1 for 1 h and then xylene alone for 1 h);

[0081] (5) Treat overnight with xylene:paraffin = 1:1 and then perform infiltration treatment;

[0082] (6) After embedding with a paraffin embedding machine, trim the excess wax blocks and place them on a paraffin slicer for sectioning (3 - 4 μm thick);

[0083] (7) Float the sections on warm water at 40°C on a spreading machine to flatten the tissue, and lift the tissue with a glass slide.

[0084] The immunofluorescence method and steps refer to Huang Yan (2021). The paraffin sections were antigen repaired in a microwave oven, blocked with BSA incubation, and then hybridized with antibodies. Anti-PcENG2 (1:200) was added, and the sections were incubated flat in a humidified box at 4°C overnight; the slides were placed in PBS (pH = 7.4) and washed 5 times at room temperature on a shaker; after drying, 1:400 diluted CY3-labeled goat anti-rabbit IgG (H+L) was added and incubated at room temperature in the dark for 1 hour, and incubated at room temperature for 1 hour; an autofluorescence quencher was added for 5 minutes and rinsed with running water for 10 minutes; the slides were placed in 3% PBS (pH = 7.4) and washed 3 times on a decolorizing shaker for 5 minutes; after standing at room temperature for 5 minutes to dry, the slides were sealed with anti-fluorescence quenching sealing agent, and photographed under a microscope.

[0085] 2.5 Protein exocytosis detection

[0086] (1) The PcENG2 signal peptide was cloned into the pSUC2 vector, and the correctly sequenced plasmid was introduced into YTK12 yeast competent cells;

[0087] (2) pSUC2-PcENG2 and pSUC2-Avr1b were added to SD-Trp medium, and the untransformed YTK12 yeast strain was inoculated into YPDA medium as a control and cultured at 30°C with shaking for 24 h;

[0088] (3) After centrifugation and removal of the supernatant, ddH2O was used to suspend the yeast cells;

[0089] (4) Add 250 μL of 10 mM acetic acid-sodium acetate buffer (pH = 4.7) and 500 μL of 10% sucrose solution, incubate at 37°C in a water bath for 10 min, centrifuge, take 100 μL of the supernatant and add 900 μL of 0.1% TTC (diluted with 1 M NaOH);

[0090] (5) Let stand at room temperature for 1-5 minutes and record the color change.

[0091] 2.6 Establishment of virus-mediated maize ZmCHI silencing (HIGS) system

[0092] The coffee nematode PcENG2 sequence was input into the VIGS fragment design website (http: / / vigs.solgenomics.net / ) for analysis and screening of suitable sequence fragments. The pTRV2 vector was linearized by double restriction endonuclease, and the target fragment was purified and recovered. The VIGS fragment was inserted into the TRV vector using a seamless ligation kit, and DH5α Escherichia coli was transformed and positive clones were selected to extract plasmids and sequenced for use.

[0093] (1) Vector linearization

[0094] The pTRV2 plasmid was digested with EcoRΙ and BamHΙ, and the reaction system is shown in Table 3.

[0095] Table 3

[0096]

[0097] Leave it standing in a 37°C water bath for 5 - 8 h, gently mix it during this period, and store it in a -20°C refrigerator after the digestion ends.

[0098] (2) Vector construction

[0099] Use specific primers with homologous arms to amplify the sample cDNA template to obtain a DNA fragment with a vector adapter, seamlessly ligate the DNA fragment with the linearized pTRV2 vector to obtain a recombinant expression vector, and transform the recombinant expression vector with correct sequencing into Escherichia coli and Agrobacterium tumefaciens C58C1 competent cells. Empty pTRV1 and pTRV2 Agrobacterium are used as blank controls, and pTRV2-ZmPDS is used as a positive control.

[0100] (3) Detection of the silencing efficiency of PcENG2 in Pratylenchus coffeae and determination of its pathogenicity

[0101] Inoculate the Pratylenchus coffeae suspension on the roots of maize at the three-leaf and one-heart stage after Agrobacterium infection. After 15 d, take out the maize roots, wash them clean, and use the Trizol liquid nitrogen grinding method to extract the RNA of maize roots in different treatment groups to detect the silencing efficiency of the target gene. There are at least 20 maize plants in each treatment group. After 60 d of inoculation, count the growth of maize (root weight, shoot weight, and rhizosphere worm population), and stain the maize roots with acid fuchsin.

[0102] 2.7 Screening of the PcENG2 - maize interaction library and yeast two-hybrid verification

[0103] Use EcoRI and BamHI to linearize the pGBKT7 vector and construct the pGBKT7-PcENG2 recombinant vector. Use the plasmid pGBKT7-Lam and pGADT7-T recombinant plasmids as negative controls, and the plasmid pGBKT7-53 and pGADT7-T recombinant plasmids as positive controls to screen for interacting proteins in the nematode-infected maize yeast library. The method for preparing yeast competent cells refers to the operation instructions of the kit of OE Biotech Co., Ltd.

[0104] (1) Plasmid co-transformation:

[0105] Add the following components to every 600 μL of yeast competent cells and vortex to mix evenly: 10 μL of maize cDNA library, 5 μg of BD recombinant plasmid, and 20 μL of denatured salmon sperm DNA.

[0106] 1) Add 2.5 mL of PEG / LiAc (250 μL of 10×LiAc, 2 μL of 50% PEG3350, 250 μL of ddH2O) to each tube and vortex to mix well;

[0107] 2) Incubate at 30 °C for 45 min, mixing every 15 min;

[0108] 3) Add 160 μL of DMSO and mix well, then incubate in a water bath at 42 °C for 20 min, mixing every 10 min;

[0109] 4) Centrifuge at 1000 rpm for 5 min, discard the supernatant, and add 3 mL of YPD plus liquid medium;

[0110] 5) Incubate with shaking at 30 °C and 250 rpm for 2 h;

[0111] 6) Centrifuge at 1000 rpm for 5 min, discard the supernatant, add 6 mL of 0.9% sterile NaCl solution, and resuspend the cells;

[0112] 7) Spread the transformed bacterial solution on a triple - deficient medium (SD / -His / -Leu), and spread 6 - 10 solid medium plates;

[0113] 8) Incubate in an incubator at 30 °C for 5 - 8 d, select yeast monoclonal colonies with a diameter greater than 2 mm, and perform PCR detection and sequencing after purification.

[0114] 2.8 Interaction verification

[0115] (1) Agrobacterium infiltration of Nicotiana benthamiana

[0116] 1) Transform the recombinant vector into Agrobacterium tumefaciens GV3101 (pSoup19) and culture in an incubator at 28 °C;

[0117] 2) Select the correct monoclonal Agrobacterium and shake it overnight;

[0118] 3) Centrifuge at 2000 rpm for 5 min at room temperature, discard the supernatant, and suspend with MMA solution and vortex to mix well until OD 600 = 0.5 - 0.8;

[0119] 4) Infiltrate the back of Nicotiana benthamiana leaves at the four - leaf stage;

[0120] 5) Observe and take pictures with a confocal microscope 2 d later.

[0121] (2) Subcellular localization detection

[0122] 1) Streak the Agrobacterium strain on an LB plate with Kan + and Rif resistance and incubate it upside - down at 28 °C for 3 d;

[0123] 2) Use a sterile pipette tip to pick up Agrobacterium and inoculate it into 5 mL of LB medium (with Kan + and Rif resistance), and culture it overnight with shaking at 28°C;

[0124] 3) Centrifuge at 2000 rpm for 5 min, discard the supernatant, and resuspend the pellet in MMA solution (10 mM MgCl2, 10 mM MES, and 0.1 mM acetosyringone), then vortex to mix well until OD 600 = 0.8 - 1.0;

[0125] 4) Let it stand at room temperature for 3 h, use a sterile needleless syringe to inject the back of the leaf, and punch holes with a puncher for detection 2 days later.

[0126] (3) Prokaryotic expression

[0127] 1) Introduce the successfully constructed pet32a and pGEX-4T-1 recombinant expression vector plasmids into BL21 Escherichia coli competent cells;

[0128] 2) Use a sterile toothpick to transfer a monoclonal bacterium into a 50 mL sterile centrifuge tube containing 10 mL of LB liquid medium (50 μg / mL ampicillin), and culture it overnight with shaking at 37°C until OD 600 = 0.5 - 0.8;

[0129] 3) Add 1 mM IPTG and culture it overnight with shaking at 20°C for 5 h;

[0130] 4) Centrifuge at 5000 rpm for 5 min, add the cell lysis solution, let it stand at room temperature for 10 min to lyse until the liquid becomes clear;

[0131] 5) Centrifuge at 8000 g for 5 min (at 4°C), collect the supernatant and precipitate separately, denature them, and then perform SDS-PAGE detection.

[0132] (4) GST pull-down

[0133] After purifying the GST-ZmCHI bait protein sample, add it to the prepared GST-tagged protein purification magnetic beads, invert and mix well, then place the centrifuge tube on a rotating mixer and incubate overnight at 4°C in a refrigerator.

[0134] 1) Washing to remove impurities

[0135] Place the centrifuge tube on a magnetic separator and let it stand for 1 min to separate the magnetic beads, add 4 times the volume of the washing solution (PBS, pH = 7.4), and then repeatedly pipette to remove impurities. Repeat the above steps 3 times.

[0136] 2) Binding of the target protein to the bait protein-magnetic bead complex

[0137] Add the purified HIS-PcENG2 sample to the prepared GST-ZmCHI complex and mix well by inverting. Place the centrifuge tube on a rotary mixer and incubate overnight at 4°C in a refrigerator (the time can be adjusted according to the binding effect).

[0138] 3) Washing away impurities

[0139] After HIS-PcENG2 binds to GST-ZmCHI, completely denature the protein sample and store it at -20°C for WB detection.

[0140] 4) Elution of the target protein

[0141] Add an eluent with a volume three times that of the magnetic beads to the above centrifuge tube, pipette 5 times, then place it on a rotary mixer at room temperature for 5 - 10 min, then place it on a magnetic separator and let it stand for 1 min. After the solution becomes clear, aspirate the supernatant and collect the elution fraction to obtain the target protein and target protein complex, which is completely denatured and then subjected to WB detection.

[0142] (5) Verification of in vivo interaction by luciferase complementation imaging

[0143] Design specific primers to amplify the PcENG2 and ZmCHI coding sequences, and insert them into the pCambia1300-cLuc and pCambia1300-nLuc vectors respectively to obtain the PcENG2-cLuc and ZmCHI-nLuc recombinant plasmids. The positive control (SAR-cLuc and SGF-nLuc plasmids) is obtained by the same method. Transform these plasmids into Agrobacterium tumefaciens GV3101 competent cells respectively. After culture and induction, mix the Agrobacterium tumefaciens carrying PcENG2-cLuc with the Agrobacterium tumefaciens carrying ZmCHI-nLuc and SGF-nLuc in equal amounts. Infiltrate the leaves of Nicotiana benthamiana with the mixed Agrobacterium tumefaciens culture alone. After treatment in an artificial climate chamber at 20°C for 48 h, infiltrate the leaves with 0.2 mM luciferin solution again and take pictures to detect luciferase activity. PcENG2-cLuc and SGF-nLuc are used as negative controls.

[0144] 2.9 Detection of plant ROS and programmed cell death

[0145] (1) Detection of plant ROS by luminol chemiluminescence method

[0146] Using a microplate reader, the reactive oxygen species (ROS) induced by flg22 in Nicotiana benthamiana were measured by the luminol chemiluminescence assay. The operation steps are as follows: Agrobacterium tumefaciens carrying the PcENG2 recombinant expression vector was infiltrated into the leaves of Nicotiana benthamiana that had grown for 4 weeks. After 2 days, circular tobacco leaves were collected using a sterile punch and placed in a microplate. 100 μL of sterile water was added, and the samples were treated in the dark at room temperature for 15 h. The sterile water in the microplate was discarded, and 100 μL of the luminol chemiluminescence mixture stored at low temperature was added on ice. The samples were placed in a microplate reader to detect their chemiluminescence values, and the dynamic changes in the ROS values were analyzed.

[0147] (2) Detection of apoptosis by Annexin V probe method

[0148] 1) Agrobacterium tumefaciens GV3101 was infiltrated into the back of the leaves of Nicotiana benthamiana that had grown for 4 weeks, and marks were made.

[0149] 2) After 24 h, 500 μL of 1× Binding Buffer (20 μL Annexin V-FITC, 40 μL PI Staining Solution) was injected into the back using a syringe.

[0150] 3) The samples were left to stand at room temperature in the dark for 10 - 15 min.

[0151] 4) After sampling the tobacco leaves, they were placed on ice and observed by fluorescence microscopy as quickly as possible.

[0152] 2.10 Extraction and purification of ZmCHI protein

[0153] (1) Extraction and purification of plant proteins

[0154] Plant proteins were extracted and purified using His-tag and GST-tag purification gels according to the instructions. The salts in the eluted proteins were removed through a Poly-Prep adsorption column (Bio-Rad Laboratories, Nevada, USA).

[0155] 2.11 Effect of ZmCHI on the egg hatching of Pratylenchus coffeae

[0156] Collect the eggs of Pratylenchus coffeae from the carrot medium, rinse them 5 times with sterile water, use a suspension containing 1000 eggs to detect the effect of ZmCHI on the eggs, incubate it with in vitro expressed ZmCHI in a 1.5 mL centrifuge tube at 25 °C, and use 1×PBS as a blank control. To detect the ovicidal activity of ZmCHI, heat ZmCHI (100 °C) for 10 min and then ice-bath for 2 min, treat the nematode eggs for 24 h. After rinsing the eggs treated differently with sterile water, place them in an incubator at 25 °C in the dark for incubation and hatching, and detect the hatching rate of the eggs treated differently under a stereomicroscope. The protein concentration was detected by the BCA method to detect the concentration of GST-ZmCHI expressed in vitro. Dilute GST-ZmCHI at an appropriate multiple, detect the maximum absorbance value at 562 nm by the enzyme-linked immunosorbent assay (ELISA) method, and calculate the protein concentration accordingly. The specific operation steps refer to the kit instructions.

[0157] 2.12 Scanning electron microscopy detection of Pratylenchus coffeae eggs

[0158] (1) Sample fixation: Under a stereomicroscope, pick the eggs of Pratylenchus coffeae with intact surfaces using eyelashes, and wash the plant tissues on their surfaces with sterile water, repeating the washing three times. Then, rinse twice with physiological saline and twice with 1×PBS solution. After centrifugation, discard the supernatant, transfer the sample to an electron microscopy fixative (such as glutaraldehyde solution), fix it at room temperature for 1 h, and then transfer it to a constant temperature at 4 °C for 12 h;

[0159] (2) Secondary fixation: The fixed sample is rinsed 2 to 5 times with 0.1 M PBS (pH = 7.4), 15 - 30 min each time. Then, use a 1% osmium tetroxide solution prepared with 0.1 M phosphate buffer (pH = 7.4) to fix it in the dark at room temperature for 1 - 2 h. Then rinse three times with 0.1 M PBS, 15 min each time;

[0160] (3) Gradient dehydration of the sample with different concentrations of ethanol and treatment with isoamyl acetate for 30 min;

[0161] (4) After drying the sample in a dryer, place it in an ion sputtering instrument to sputter gold for 40 s, and then observe and save the image using a scanning electron microscope.

[0162] 2.13 Real-time fluorescence quantitative RT-qPCR analysis of differential expression of defense genes

[0163] The differential expression of defense genes in Nicotiana benthamiana transiently expressing PcENG2 in response to flg22-induced immune responses was detected by RT-qPCR, and the effects of PcENG2 transient expression on the transcriptional levels of tobacco defense-related genes NbPR1, NbPAL, and NbLOX were detected. The Agrobacterium infiltration method was used to perform microextraction and reverse transcription of RNA from the leaves of Nicotiana benthamiana transiently expressing PcENG2-GFP and treated with flg22. RT-qPCR was used to detect the expression of defense-related genes, with tobacco NbEF1 as the internal reference.

[0164] 2.14 Data processing and analysis

[0165] In this example, the significance analysis of two groups of data was performed using an independent samples T-test (Student's Test, abbreviated as T-test), and the significance difference analysis of multiple groups of data was performed using analysis of variance (Analysis of Variance, abbreviated as ANOVA). The independent samples T-test is commonly used to compare whether the means of a certain variable in two independent samples are significantly the same. Common types of analysis of variance include the comparison of the means of multiple samples grouped by a single factor and the comparison of the means of multiple samples grouped by two factors.

[0166] 3 Results and analysis

[0167] 3.1 The infectivity and pathogenicity of Pratylenchus coffeae decreased significantly after PcENG2 gene silencing

[0168]

[0169] Maize plants transformed with the empty pTRV2 plasmid and pTRV2-GFP were used as negative controls, and maize plants transformed with pTRV-ZmPDS were used as positive controls. After infecting maize seeds with Agrobacterium, it was found that obvious "photobleaching" occurred in the germinated maize plants transformed with pTRV-ZmPDS, while no obvious growth defect characteristics were observed in the negative control maize plants( Figure 1 ).

[0170] The roots of maize plants in the silencing treatment group infected with Pratylenchus coffeae were stained with acid fuchsin. The results showed that a large number of Pratylenchus coffeae were distributed in the roots of the pTRV2, pTRV2-GFP, and wild-type (WT) maize treatment groups( Figure 2 A-C in), and the number of nematodes in the roots of maize plants in the pTRV2-PcENG2 treatment group was significantly reduced( Figure 2 D in).

[0171] After measuring the growth of maize plants in different treatment groups, it was found that the expression level of PcENG2 decreased significantly by about 56%( Figure 3 A in). The results at 60 dpi showed that the number of P. coffeae in the rhizosphere of pTRV2-PcENG2 maize was significantly reduced( Figure 3 B in), and the fresh weight of the aboveground parts and roots of maize plants in the pTRV2-PcENG2 treatment group increased significantly( Figure 3 C, D in).

[0172] 3.2 Immunolocalization of Pratylenchus coffeae PcENG2 in Maize Roots

[0173] To detect the distribution of PcENG2 in nematodes and maize roots, the specific protein sequence of PcENG2 was expressed prokaryotically to prepare a polyclonal antibody against PcENG2 in rabbits. The results of Western blot detection showed a signal band at the expected 37 kDa( Figure 4 A in). After diluting the antibody at a dilution ratio of 1:1000, 10 ng, 5 ng, 1 ng, and 500 pg antigens were detected respectively, and the antibody had high detection sensitivity( Figure 4 B in).

[0174] The purified specific antibody was used to detect the sections of maize roots infected with Pratylenchus coffeae. The immunolocalization results showed that PcENG2 was aggregated at the site of nematode infection( Figure 5 A, B in), and immunoscanning observed that the PcENG2 immune signal was distributed around the infection path of Pratylenchus coffeae( Figure 6 A, B in). Using pre-immune serum( Figure 5 C in) and hybridization with CY3-labeled secondary antibody( Figure 5No immune signal was detected in D), and the research shows that PcENG2 is released during the invasion of host cells by Pratylenchus coffeae.

[0175] 3.3 Verification of the excretory property of the PcENG2 signal peptide

[0176] Using the online bioinformatics analysis websites SMART and SignalP 4.0 to predict the structure of the PcENG2 signal peptide, a 22aa signal peptide sequence ( Figure 7 ) was found. The recombinant vector (pSUC2-PcENG2-SP) was constructed by ligating the 22aa signal peptide sequence encoding the PcENG2 protein with the pSUC2 vector, and then transformed into the YTK12 yeast strain to detect whether invertase could be secreted. Avr1b-SP was used as a positive control. The growth test on the synthetic tryptophan (Trp)-deficient agar medium showed that PcENG2-SP and Avr1b-SP grew normally on the sucrose medium ( Figure 8 ), and YTK12 yeast (PcENG2-SP) secreted invertase, reducing 2,3,5-triphenyltetrazolium chloride (TTC) to the red insoluble triphenylformazan (TTF). Therefore, the PcENG2 signal peptide has excretory property.

[0177] 3.4 PcENG2 inhibits plant ROS and programmed cell death

[0178] The Agrobacterium tumefaciens competent cells carrying PcENG2-GFP, green fluorescent protein (GFP), and ZmCHI-GFP were transformed, and after the strain was propagated and cultured, the 4-week-old Nicotiana benthamiana leaves were infiltrated for transient expression. After 48 hours of infiltration with Agrobacterium, the round leaves of Nicotiana benthamiana were punched and collected, and after treatment with 100 nM flg22, the ROS was measured by an enzyme-labeling instrument. After treating Nicotiana tabacum with H2O + flg22, the ROS value showed an increasing trend at the initial stage and gradually decreased after reaching the peak; the ROS value of the GFP + flg22 treatment group also showed an initial increase to the peak and then gradually decreased; the ROS value of the PcENG2 + flg22 treatment group increased slightly at the initial stage to the peak and then gradually decreased. Compared with the ROS peak of the control group, the peak value was significantly reduced. The results show that the transient expression of PcENG2 in Nicotiana benthamiana significantly inhibits the ROS burst in Nicotiana benthamiana ( Figure 9 ).

[0179] Prepare the Agrobacterium strain mixture of Gpa2 / RBP-1 and the Agrobacterium strain GV3101 carrying the recombinant expression vectors of PcENG2 and ZmCHI. After propagation and culture, infiltrate the leaves of Nicotiana benthamiana at 4 weeks old simultaneously. 48 hours after Agrobacterium infiltration, infiltrate the Annexin V-FITC and PI Staining Solution on the back of the transiently expressed Nicotiana benthamiana leaves. After treatment at room temperature in the dark, it was found that Gpa2 / RBP-1 could induce programmed cell death in Nicotiana benthamiana cells, and there was no fluorescence signal in the transient expression of PcENG2 in Nicotiana benthamiana, indicating that the transient expression of PcENG2 inhibited the programmed cell death induced by Gpa2 / RBP-1( Figure 10 ).

[0180] 3.5 PcENG2 inhibits the expression of host defense genes

[0181] Transiently express PcENG2 in Nicotiana benthamiana leaves and detect its effect on tobacco defense-related genes (NbCAT, NbPAL, NbPR1, and NbLOX) stimulated by flg22. By analyzing the transcriptional levels of defense-related genes, it was found that the expression levels of NbPAL and NbPR1 genes in the SA pathway of tobacco were significantly down-regulated, and the expression level of the NbLOX gene in the JA pathway was significantly up-regulated( Figure 11 ). Therefore, PcENG2 affects the expression of host defense-related genes.

[0182] 3.6 Screening of interacting proteins in the PcENG2 yeast two-hybrid library

[0183] Co-transform the constructed BD-PcENG2 plasmid with the secondary library plasmid into yeast Y2H competent cells. White yeast single colonies appeared after 3 days. Pick out the yeast colonies on the screening medium plate and transfer them to a liquid medium (SD / -Leu / -Trp / -His) for propagation and culture. Then, pipette 2 μL, dilute it 100 times with sterile water, and spot it on the plate of SD / -Trp / -Leu / -His / -Ade-X-α-Gal to screen for blue colonies( Figure 12 in A) for PCR bacterial liquid detection( Figure 12 in B) to obtain the sequence information of candidate interacting proteins. Sequencing and alignment analysis of positive monoclonal clones found that three positive clones might have an interaction relationship with PcENG2. The proteins were: maize chitinase, cysteine protease, and chloroplast envelope quinone oxidoreductase (Table 4).

[0184] Table 4 PcENG2 candidate interacting proteins

[0185]

[0186] 3.7 Verification of the one-to-one interaction between PcENG2 and ZmCHI

[0187] The amino acid sequence of PcENG2 protein is shown in SEQ ID NO.2: MSAFNLLSVLFSLTSLAYGANPPYGKLSLSAAQLVGSSNQAVQLRGMSFYWSQWDNPDFWTAAVVKALACNWNANVVRAAMAVESAYGGYLSGGSTATTQLNKVYTVADAAIANGIYVLIDWHETGNTAYTSQAVSFFSTVSKKYANVPNVLYEIWNEPTNEYTDWATVRNYHMAVIKAIRANDANAIIIAGTPKWSSGIDSNVLANPITGYKNVMYTLHWYPNGAAWQQYQRDSITTAKKKGLATFITEYGVASDANTAVNASESNLWWTYLDNNKVSYINWHVGSISESWSILKSGTQPSQITSDSLLTDSGKLVKQKLKSVSNGVACFSG;

[0188] The ZmCHI protein sequence is shown in SEQ ID NO.3: MAANLKWAPVLALVVVVAAMVGTTSAGNIAVYW GQNGNEGSLADACNSGLYAYVNIAFLTTFGNGQTPVLNLAGHCDPGSGSCTGQSSDIQTCQSLGIKVLLSIGGASGSYGLSSTDDANSVADYLWDNFLGGSGSSRPLGAAVLDGIDFDIENGQSAHYDDLANALKGKGSVLLTAAPQCPYPDASLGPALQTGQFDNVWIQFYNNPGCAYANGDDTNLVNAWNTWTSSITAGSFYLGVPASPQAAGSGYIDPGTLTGTVIPAIRGIGNYGGIMVWDRFNDVQNNYSSQVKGSV。

[0189] Yeast cells carrying the bait protein (pGBKT7-PcENG2) and the prey protein (pGADT7-ZmCHI) grew normally on SD / -Trp / -Leu (SD-WL) medium. After adding 1 mg / mL Aureobasidin A and 20 mg / mL X-α-Gal, the yeast cells grew blue on the selective quadruple-deficient medium (SD / -Trp / -Leu / -His / -Ade, SD-WLHA), AD-ZmCHI+BD-PcENG2 235-289aa and AD-ZmCHI+BD-PcENG2 193-234aaThe SD-WLHA turned blue, indicating that there was an interaction between the cellulase and chitin-binding protein domains of the PcENG2 protein and ZmCHI( Figure 13 ). Yeast cells containing p53 and T antigen were used as positive controls, and yeast cells containing Lam and T antigen were used as negative controls.

[0190] 3.8 Pull-down verification of the interaction between PcENG2 and ZmCHI

[0191] GST Pull-down is an effective method for verifying protein-protein interactions in vitro. By constructing PET-32a and PGEX-4T-1 vectors and transforming BL21(DE3) Escherichia coli competent cells, the PcENG2-His protein and ZmCHI-GST were expressed prokaryotically, with PcENG2-His and GST as negative controls.

[0192] The results showed that when using antibody-HIS and antibody-GST antibodies to detect the proteins captured by GST magnetic beads, it was found that His-PcENG2 could be captured by GST magnetic beads that had adsorbed ZmCHI-GST, but not by GST protein( Figure 14 ).

[0193] 3.9 Luciferase complementation verification of the interaction between PcENG2 and ZmCHI in vivo

[0194] The Agrobacterium cultures carrying PcENG2-nLuc or cLuc-ZmCHI were mixed (cLuc-ZmCHI + PcENG2-nLuc) and infiltrated into Nicotiana benthamiana leaves. The area of Nicotiana benthamiana leaves infiltrated with Agrobacterium expressing SGF-nLuc + cLuc-SAR was used as a positive control, and SGF-nLuc + cLuc-ZmCHI was used as a negative control. The interaction between PcENG2 and ZmCHI was verified by luciferase complementation assay.

[0195] The test results showed that fluorescence signals could be observed after co-expression of PcENG2-nLuc and cLuc-ZmCHI in Nicotiana benthamiana leaves 2 days after Agrobacterium infiltration, and no fluorescence signal was observed after co-expression of cLuc-ZmCHI and nLuc( Figure 15 ). LCA further indicated that there was an interaction between PcENG2 and ZmCHI. LCI was measured in tobacco leaves to confirm the interaction between cLuc-ZmCHI and PcENG2-nLuc.

[0196] 3.10 BIFC verification of the interaction between PcENG2 and ZmCHI

[0197] The PcENG2 expression sequence was ligated to the N-terminus of nYFP to construct a recombinant expression vector, and ZmCHI was ligated to the C-terminus of nYFP to construct a recombinant expression vector. Agrobacterium carrying nYFP-PcENG2 and nYFP-ZmCHI was mixed in equal volumes and infiltrated into Nicotiana benthamiana leaves for transient expression to verify the interaction relationship. As Figure 16 shown, there was no yellow fluorescence in the control group, and yellow fluorescence appeared in the cytoplasm and nucleus of nYFP-PcENG2 + nYFP-ZmCHI. The research shows that PcENG2 and ZmCHI interact in the cytoplasm and nucleoplasm.

[0198] 3.11 Silencing of maize ZmCHI promotes nematode infection

[0199] The nucleotide sequence of the ZmCHI gene is shown in SEQ ID NO.4: ATGGCGGCTAATCTCAAGTGGGCTCCTGTTCTAGCCCTCGTCGTCGTCGTTGCTGCCATGGTTGGCACCACGAGCGCCGGCAACATCGCAGTGTACTGGGGCCAGAACGGCAACGAAGGCAGCCTGGCGGACGCCTGCAACTCCGGGCTCTACGCCTACGTCAACATCGCGTTCCTCACAACCTTCGGCAACGGACAGACCCCAGTCCTCAACCTCGCCGGACACTGCGACCCCGGCTCTGGCAGCTGCACCGGCCAGAGCAGCGACATCCAGACCTGCCAGTCGCTGGGCATCAAGGTGCTCCTCTCCATCGGCGGCGCCAGCGGCAGCTACGGCCTCTCCTCCACCGACGACGCCAACAGCGTGGCCGACTACCTGTGGGACAACTTCCTGGGCGGCAGCGGCTCGTCCCGCCCGCTCGGCGCCGCCGTGCTCGACGGCATCGACTTCGACATCGAGAACGGCCAGTCGGCGCACTACGATGACCTGGCGAACGCGCTCAAGGGCAAGGGGAGCGTGCTGCTGACGGCGGCGCCGCAGTGCCCCTACCCGGACGCGTCGCTGGGGCCGGCGCTGCAGACGGGCCAGTTCGACAACGTGTGGATACAGTTCTACAACAACCCCGGGTGCGCGTACGCCAACGGGGACGACACCAACCTGGTGAACGCATGGAACACGTGGACCAGCAGCATCACCGCGGGGAGCTTCTACCTCGGCGTCCCAGCGTCTCCCCAGGCCGCGGGCAGCGGGTACATCGACCCCGGCACCCTGACGGGCACGGTGATCCCCGCCATCCGAGGCATTGGCAACTACGGCGGCATCATGGTGTGGGACCGCTTTAACGACGTGCAGAACAACTACAGCAGCCAGGTGAAGGGCAGCGTCTGA;

[0200] Virus-mediated RNAi-ZmCHI in maize showed that the resistance of RNAi-ZmCHI maize plants to Pratylenchus coffeae was weakened. After transforming maize with Agrobacterium tumefaciens carrying TRV-ZmCHI, the expression level of ZmCHI decreased significantly by about 45% ( Figure 17 in A). Statistical results of the experiment after inoculating nematodes on maize roots in different treatment groups for 60 days showed that the number of nematodes infecting the rhizosphere of RNAi-ZmCHI maize plants increased by about 13% ( Figure 17 in B), and the growth parameters such as the fresh weight of the above-ground part and root weight of maize decreased significantly ( Figure 17 in C, D).

[0201] Pratylenchus coffeae, Pratylenchus zeae, Pratylenchus scribneri, and Pratylenchus penetrans were inoculated on the roots of RNAi-ZmCHI maize respectively. The number of nematodes in the maize rhizosphere was counted 60 days after inoculation. The results of inoculating Pratylenchus coffeae showed that the number of nematodes infecting the rhizosphere of RNAi-CHI maize plants increased significantly ( Figure 18 ). Therefore, maize ZmCHI can be used as one of the control targets for broad-spectrum resistance to Pratylenchus nematodes.

[0202] The inventors constructed maize plants overexpressing ZmCHI, using wild-type maize plants as the control group. The results showed that the expression level of ZmCHI increased significantly. Statistical results of the experiment after inoculating nematodes on maize roots in different treatment groups for 60 days showed that the number of nematodes infecting the rhizosphere of ZmCHI overexpressing maize plants decreased, and the growth parameters such as the fresh weight of the above-ground part and root weight of maize increased significantly, and the resistance of maize to Pratylenchus coffeae increased significantly. Therefore, maize ZmCHI plays an important role in inhibiting nematode infection and enhancing host resistance.

[0203] 3.12 ZmCHI inhibits the hatching of nematode eggs

[0204] The in vitro expressed ZmCHI-GST was enriched by gel purification and then used to treat the eggs of Pratylenchus coffeae washed from the carrot medium. Eggs treated with 1×PBS, ZmCHI-GST, and ZmCHI-GST and PcENG2-His co-incubated for 24 h were photographed by scanning electron microscopy.

[0205] Scanning electron microscopy images showed that the ZmCHI-GST protein affected the morphological development of Pratylenchus coffeae eggs. The surface of the normally developing eggs was smooth and plump without rupture. In contrast, after treating the eggs with ZmCHI-GST for 24 h, morphological damages such as invagination, rupture, and leakage of contents were observed on the eggshell surface ( Figure 19 ). After treating the eggs at room temperature in the dark for 24 h and 48 h respectively, the egg morphology was fixed with fixative, using 1×PBS as the blank control. It was found that ZmCHI-GST significantly inhibited the hatching of Pratylenchus coffeae eggs. The hatching rates of eggs co-incubated for 24 h and 48 h decreased by about 25.6% compared with the control group ( Figure 20), with significant differences.

[0206] 3.13 PcENG2 inhibits the activity of ZmCHI

[0207] The plasmids PET32a-PcENG2 and PGEX-4T-1-ZmCHI were respectively transformed into the BL21(DE3) Escherichia coli strain, and PcENG2-HIS and ZmCHI-GST were induced to express in vitro to detect the effect of PcENG2-HIS on the enzyme activity of ZmCHI-GST, with 1×PBS solution as the blank control.

[0208] The results showed that PcENG2-HIS reduced the activity of ZmCHI, and PcENG2-HIS after heat denaturation treatment had no significant effect on the enzyme activity of ZmCHI-GST ( Figure 21 ). The research shows that PcENG2 can significantly inhibit the activity of ZmCHI.

[0209] In summary, through RNAi-PcENG2, the present invention clarified that PcENG2 is involved in the infection of nematodes. In the immunolocalization analysis of PcENG2 of Pratylenchus coffeae in maize roots, it was found that PcENG2 was abundantly distributed near the migration path of nematodes. The transient expression of PcENG2 in Nicotiana benthamiana can inhibit the expression of host defense genes, inhibit the ROS burst and host cell programmed death induced by flg22. In order to clarify the interaction mode between PcENG2 and the host, through the screening and verification of yeast library interacting proteins, it was found that ZmCHI interacts with PcENG2. Further research found that the resistance to Pratylenchus coffeae decreased significantly after ZmCHI was silenced, indicating that ZmCHI is involved in the infection resistance to nematodes. In addition, PcENG2 can significantly reduce the activity of ZmCHI and inhibit ZmCHI from degrading the body wall and eggshell of nematode eggs. ZmCHI has broad-spectrum resistance to Pratylenchus nematodes and may be a potential control target for Pratylenchus nematodes.

[0210] Obviously, the above-mentioned embodiments of the present invention are only examples for clearly explaining the present invention, and are not intended to limit the implementation manner of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. Application of maize ZmCHI gene in regulating plant resistance to Pratylenchus nematodes, characterized in that: The nucleotide sequence of the ZmCHI gene is shown in SEQ ID NO.

4.

2. The use according to claim 1, characterized in that: Silencing or knocking out the ZmCHI gene reduces the plant's resistance to Pratylenchus nematodes.

3. The use according to claim 1, characterized in that: The plants include corn.

4. The use according to claim 1, characterized in that: The short-bodied nematodes include coffee short-bodied nematodes, corn short-bodied nematodes, scribner short-bodied nematodes and sonian short-bodied nematodes.

5. A method for improving plant resistance to Pratylenchus nematodes, characterized in that: By overexpressing the ZmCHI gene in a plant, the plant's resistance to short-bodied nematodes is improved.

6. The method according to claim 5, characterized in that The method for overexpressing the ZmCHI gene in the plant is selected from at least one of the following methods: 1) By introducing a plasmid with the ZmCHI gene; 2) by increasing the copy number of the ZmCHI gene on plant chromosomes; 3) by changing the promoter sequence of the ZmCHI gene on the plant chromosome; 4) by operably linking a strong promoter to the ZmCHI gene; 5) By introducing enhancers.

7. Use of a recombinant vector, expression cassette or transgenic cell line containing the ZmCHI gene in improving plant resistance to Brachymemma nematodes.

8. Application of ZmCHI protein in regulating plant resistance to Pratylenchus nematodes, characterized in that: The amino acid sequence of the ZmCHI protein is shown in SEQ ID NO.

3.

9. Application of ZmCHI protein in inhibiting the hatching of coffee nematode eggs.

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